Tumor Necrosis factor-related apoptosis-inducing ligand (TRAIL) is regarded as a potential anti-cancer agent, however, considerable numbers of cancer cells, especially glioblastoma multiforme (GBM), are resistant to apoptosis induction by TRAIL. The aim of this proposal is to evaluate the naturally secreted Gaussia Luciferase (Gluc) as a reporter for high throughput drug screening in order to identify small molecules that sensitize glioma cells to TRAIL. Different glioma cells will be engineered by gene transfer to express Gluc which expression, and therefore cell viability, can be monitored over time by subjecting an aliquot of the conditioned medium to bioluminescence measurements using a 96-wells format plate luminometer. Initially, different components of the Gluc assay including well-to-well variation, as well as substrate stability over time and optimal substrate dose will be optimized for a 96-well plate format. Next, different glioma cell lines, including primary GBM cells dissociated from patient tumor sections, will be tested for their sensitivity or resistance to TRAIL. Finally, primary GBM cells will be screened for drugs that will sensitize them to TRAIL using small molecule libraries some of which are FDA-approved drugs known to cross the blood-brain barrier. The drug candidates that sensitize primary GBM cells to TRAIL will be further characterized in dose- and time-dependent experiments in vitro and potentially in our experimental glioma-Gluc models, in vivo. The Gluc assay time of 4 seconds makes it a valuable tool for HTS and can be easily applied into 384-well plate format to screen thousands of compounds over a relatively short period of time.